Purification and Characterization of a 43-kDa Rotenone-insensitive NADH Dehydrogenase from Plant Mitochondria

doi:10.1074/jbc.271.38.23117

Purification and Characterization of a 43-kDa Rotenone-insensitive NADH Dehydrogenase from Plant Mitochondria*

R. Ian Menz^‡ and
David A. Day^§

From the Division of Biochemistry and Molecular Biology and the Co-operative Research Centre for Plant Science, The Australian National University, Canberra, ACT 0200, Australia

^§ To whom correspondence should be addressed:
Division of Biochemistry and Molecular Biology, The Australian National University, Canberra, ACT 0200, Australia.
Tel.: 61-6-2492870; Fax: 61-6-249-0313; E-mail david.day{at}anu.edu.au

Abstract

A 43-kDa NAD(P)H dehydrogenase was purified from red beetroot mitochondria. An antibody against this dehydrogenase was used in conjunction with the membrane-impermeable protein cross-linker 3,3′-dithiobis(sulfosuccinimidylpropionate) to localize the dehydrogenase on the matrix side of the inner membrane. Immunoblotting showed that the dehydrogenase was found in mitochondria isolated from several plant species but not from rat livers. Antibodies against the purified dehydrogenase partially inhibited rotenoneinsensitive internal NADH oxidation by inside-out submitochondrial particles. The level of rotenone-insensitive respiration with NAD-linked substrates correlated with the amount of 43-kDa NAD(P)H dehydrogenase present in mitochondria isolated from different soybean tissues. Based on these results, we conclude that the 43-kDa NAD(P)H dehydrogenase is responsible for rotenone-insensitive internal NADH oxidation in plant mitochondria.

Footnotes

↵^‡ Recipient of a graduate student assistantship from the Co-operative Research Center for Plant Science.
↵* This work was supported by an Australian Research Council grant (to D. A. D.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SMP

submitochondrial particles

PAGE

polyacrylamide gel electrophoresis

DTSSP

3-3′-dithiobis(sulfosuccinimidylpropionate)

deamino-NADH

nicotinamide hypoxanthine dinucleotide, reduced form

E64

trans-epoxysuccinly--luecylamido-(4-guanidino)butane; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane

Q₀

2,3-dimethoxy-5-methyl-1,4-benzoquinone

FeCN

potassium ferricyanide

mersalyl

o-[3-hydroxymercuri-2-methoxypropyl)carbamoyl] phenoxyacetic acid

dicumarol

3-3′methylene-bis(4-hydroxycoumarin)

flavone

2-phenyl-4H-1-benzopryan-4-one

bis-Tris

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
- Received May 8, 1996.
- Revision received June 14, 1996.